The pathway followed by secreted proteins within the cell has been studied in prokaryotes, yeast and mammalian cells. A secreted protein must first be targeted to its destination, and then must be able to cross the target organelle membrane. In recent years a great deal has been learned about the process of targeting or translocation of proteins from the cytoplasm into the endoplasmic reticulum (ER). A secretory signal peptide in the primary amino acid sequence targets the protein for secretion. The translocation process of secretory proteins into the ER, begins on the ribosomes in the cytoplasm. Once the signal peptide is synthesized and emerges from the ribosome, a cytosolic factor, the signal recognition particle, or SRP, binds to the signal peptide and to the ribosome, causing translational arrest. The nascent peptide/SRP/ribosome complex is recognized by an SRP receptor (SRP docking protein) in the ER membrane and binds to it; the signal peptide is then displaced from the SRP, in steps requiring GTP hydrolysis. Protein synthesis then continues, and the nascent protein enters the ER (Walter and Lingappa, Ann. Rev. Cell Biol. 2: 499-516, 1986; Simon, Current Opinion in Cell Biology 5: 581-588, 1993).
The SRP is a ribonucleoprotein composed of a single RNA molecule (7S RNA) and 6 polypeptides identified by their molecular weights: 9 kDa, 14 kDa, 19 kDa, 54 kDa, 68 kDa, and 72 kDa (Walter and Blobel, Nature 299: 691-698, 1982; Siegel and Walter, J. Cell Biol. 100: 1913-1921, 1989). Genes coding for the 54 kDa subunit of SRP have been cloned from higher eukaryotes (murine and canine; Bernstein et al., Nature 340: 482-486, 1989; Romisch et al. Nature 340: 478-482, 1989, respectively), Saccharomyces cerevisiae and Schizosaccaromyces pombe (Hann et al. J. Cell Biol. 109: 3223-3230); and Arabidopsis thaliana (Lindstrom et al., Plant Molecular Biology 23: 1265-1272, 1993). Analysis of these genes has revealed that the protein consists of two functional domains. Using fine resolution cross linking for nascent secretory peptide chains to SRP, it has been demonstrated that the C-terminal half of the protein binds to the signal peptide (Kurzhchalia et al., Nature 320: 634-636, 1986; Krieg et al., Proc. Nat. Acad. Sci. 83: 8604-8606, 1986). This domain is referred to as the M-domain due to its high percentage of methionine residues. The N-terminal half of the protein has been identified as a GTP binding domain, based on amino acid homologies with consensus GTP binding motifs (Bernstein et al., Nature 340: 482-486, 1989; Romisch et al. Nature 340: 478-482, 1989).
There have been no previous studies on the SRPs of filamentous fungi. Filamentous fungi are now commonly used as host cells for expression of recombinant protein, and there is an ongoing search for means to enhance levels of secretion of a protein of interest. Isolation of the genes encoding crucial units of the secretory pathway will facilitate this process. To that end, the present invention provides a gene encoding an SRP subunit, and thus also provides a means of increasing heterologous protein secretion in filamentous fungal host cells.